The overall objective of the proposed studies is to address the critical signaling pathways by which pro-inflammatory mediators such as thrombin mediate the expression of adhesion molecule ICAM-1 (CD54) in endothelial cells and thereby induce firm neutrophil (PMN) adhesion. We hypothesize that thrombin induces NF-kB activation and ICAM-1 expression by the interaction of specific heterotrimeric G-proteins with the Proteinase Activated Receptor-1 (PAR-1) and the resultant activation of phosphatidylinositol 3-kinase (PI3 kinase)-dependent signaling pathways. We further postulate that thrombin-induced expression of endothelial adhesivity by this mechanism leads to lung PMN sequestration and PMN-dependent lung vascular injury and tissue edema. To gain new insights into the molecular pathogenesis of PMN-dependent lung vascular injury, this proposal will focus on specific aspects of thrombin activation of PAR-1 on intracellular signaling which in turn regulate the activation of NF-kB and expression of ICAM-1 in endothelial cells. The specific aims of this proposal are to: 1. Determine the role of Galpha-q that is functionally coupled to PAR-1 in mediating thrombin-induced NF-kB activation and ICAM-1 expression, and resultant endothelial adhesivity and PMN migration across endothelial barrier. 2. Determine the role of PI3 kinase and the downstream signaling events in transducing PAR-1-activated signals that mediate NF-kB activation and ICAM-1 expression. 3. Address the role of Galpha-i, which is functionally coupled to PAR-1, in preventing thrombin-induced NF-kB activation and ICAM-1 expression in endothelial cells. These studies will use primary cultures of human vascular endothelial cells and mouse models. We will use pharmacological agents and recombinant adenoviruses encoding activating or dominant negative forms of signaling molecules to modulate the genetic expression of endotheial cells. Finally, we also will use PAR-1 and PI3 kinase knockout mouse models to address their in vivo role in the mechanism of thrombin-induced lung PMN uptake and PMN-dependent lung vascular injury and tissue edema. The information gained will increase our understanding of the regulation of endothelial adhesivity and provide future directions for pharmacological and genetic manipulations to interfere with inappropriate PMN sequestration and PMN-mediated lung injury.